Giebel W, Striebel H M
Microsc Acta Suppl. 1977(1):133-49.
In order to obtain a better reproducibility in quantitative determination of amino acids in amounts of picomoles an internal standard was used. A mixture of ten amino acids was diluted and dansylated. 0.1 microliter of each solution was separated by two-dimensional thin-layer chromatography on polyamide sheets (3 x 3 cm). Each chromatogram was photographed with four different exposure times under ultraviolet light. The dark spots in the photographic negatives were measured by television densitometry. A number of factors influence the reproducibility of this method: loading volume, photographic exposure time and processing, and quenching of fluorescence. As an internal standard, which is influenced by all these factors, DANS-hydroxyproline was added to each solution. By using this standard, the variations in exposure time and fluorescence quenching can be corrected satisfactorily. The relative standard deviation of the mean value of four photographs of one chromatogram is 2%. The results on correction of the volume deviation were not so satisfying. The relative standard deviation of the mean of twelve different chromatograms can be reduced from 34% for the initially measured data, to 8% for the values corrected by the internal standard. It seems necessary to carry out three or more determinations of one solution. In this case the relative standard error of the mean is 2%.
为了在皮摩尔量的氨基酸定量测定中获得更好的重现性，使用了内标。将十种氨基酸的混合物稀释并进行丹磺酰化。每种溶液取0.1微升在聚酰胺板（3×3厘米）上通过二维薄层色谱进行分离。每张色谱图在紫外光下用四种不同的曝光时间拍照。通过电视密度计测量照相底片上的暗斑。许多因素会影响该方法的重现性：进样量、照相曝光时间及冲洗过程以及荧光猝灭。作为受所有这些因素影响的内标，向每种溶液中加入丹磺酰羟脯氨酸。通过使用该标准品，可以令人满意地校正曝光时间和荧光猝灭的变化。一张色谱图的四张照片平均值的相对标准偏差为2%。体积偏差校正的结果不太令人满意。十二个不同色谱图平均值的相对标准偏差可以从最初测量数据的34%降低到经内标校正后的值的8%。对一种溶液进行三次或更多次测定似乎是必要的。在这种情况下，平均值的相对标准误差为2%。
